1. Alternative splicing in human tissues John Castle July 20, 2007 Rosetta / Merck Seattle, USA

2. Acknowledgements Rosetta/Merck Jason Johnson Chris Armour Ronghua Chen Phil Garrett-Engele Amit Kulkarni Lee Lim Chris Raymond Jyoti Shah Collaborators Tom Cooper (Baylor) (Data not shown) Guey-Shin Wang Duyen Tran Chaolin Zhang (summer intern from CSHL) Publications cited Spellman and Smith, 2006 Boutz et al., 2007 (Ares and Black labs) Zhu, B. et al., 2005 (Gulick lab)

3. Needs for alternative splicing at a pharmaceutical company Biomarker identification Identify transcript structures expressed in a tissue, including novel isoforms Report regulation of known isoforms for use in pathway analysis or as drug targets Requirements Robust, high-confidence, high-throughput profiling –Array patterns, amplification, sequencing Visualization and analysis tools for profiling data Interpretation, including isoform function and pathways

4. Discovery-mode junction arrays (Refseqs only): multiple samples help, the events you don’t monitor hinder interpretation Junction Probe X06989 NM_201414 NM_000484 Predictions from residuals (model-observed) Junction Tissues Log probe intensity Junction Melanoma Lung Carcinoma Brain Fetal Brain Brain-Amygdala 400 300 200 100 Brain samples 6795 RT-PCR Primers mixture with non-Refseq not predicted (no probe) Gene = APP

5. Some probe strategies and limitation for microarray patterns Exon arrays –Easiest to design and interpret –Miss substantial fraction of known AS events 5’SS, 3’SS, intron retentions Junction arrays –Less control over probe position –Single probe events difficult to interpret (e.g. 3-nt 3’SS vs. 300 nt inserted exon), complicating validation –More known and unknown events detected, but accordingly more difficult to interpret –May want to use exon-to-intron junction probes as controls for “half-hybridization” in intensity-based experiments Junction + exon arrays –2x expense, but more markers of same event –Different probe lengths affect intensities (not ratios) Splicing event arrays (mutually exclusive probes) –Coverage of more splicing events from ESTs –Need extra probes to estimate gene-level expression –Can’t detect novel splicing events –Unmonitored splicing events/mixtures more easily confound analysis Junction + exon + mutually exclusive probes for known events –Expensive –Most complex data analysis –Best balance of discovery and monitoring (one data set for both) –Enables reciprocal analysis All methods –False predictions created by saturated probes, dim probes, constant probes, specific & non-specific cross-hybridization –Splicing predictions can be confounded by strong gene-level regulation or complex isoform mixtures –Read-out differentially regulated splicing events – difficult to accurately quantitate isoforms –More samples help

6. Brain Fetal Brain Colon 400 300 200 100 Junction probe for double exon drop Junction + Exon + Isoform-monitoring probes 1 10 * 12 atg 1 8 * 7

7. Analysis tools for alternative splicing profiling data Confounding factors include Inaccurate estimates of the gene expression Spurious measurements, such as from cross- hybridization, bad probes, and high background Needed output A confidence value (e.g., a p-value) Expression changes, including both: –Exon regulation –Splicing event regulation (e.g., % of total gene expression) Several measurement types, including: –Novel transcript script structure –Individual regulated exon (may be a minor component) –Differentially expressed alternative splicing event

8. Synthetic data show how dark and saturated can lead to compressed ratios These probes compress ratios and can erroneous values: Probes with high background Probes always dim Probes near saturation If not filtered, these appear to be novel splicing predictions.

9. Real data showing single probe predictions with high cross-hyb potential Gene level consistent across samples, but relatively low All alternative isoforms show low expression in both samples and no regulation. One probe shows differential expression with intensities much higher than the gene expression, suggesting cross-hybridization.  We have an 0-for-8 validation rate of these predictions.

10. What transcript structure is expressed? Large prediction, but change in isoform ratios is meaningless. The RefSeq isoform (exon 17) is not expressed, just alternate form (#2). looks like a splicing prediction, but isn’t Probes with high background and constant- intensity probes create false predictions. The cDNA lacking exon 17 should be used for screening. No alternative splicing

11. Exon 10 is a novel cassette exon. The use of multiple probes increases the confidence of this prediction. The exon 10 is upregulated ~10-fold. However, the low intensities on probes monitoring exon 10 suggest the +exon 10 isoform is a minor component. A junction probe spanning exon 9-to-11 would enable a determination of the relative abundance. Example of a potential biomarker

12. Validation of alternative splicing profiling of sample pairs Eliminate suspect measurements, such as high background, from non- expressing genes, or that are not consistent with alternative splicing. Validate by RT-PCR only changes in size greater than 3-nt. Our Version 2 algorithm achieves >80% validation for call rate and has a good correlation (left). The algorithm is a reciprocal measurement, similar to Ule & Darnells and ExonHit’s, with basic additional filtering. Version 3 (being tested) includes a p-value and additional filters.

13. Interpretation of splicing changes Currently in pathways: - transcription factor to targets Need to add systems biology at a splicing level, such as: - splicing factor to target exons - isoform specific nodes in pathways - isoform specific phenotypes & functions Disease markers? Casual? Necessary? KEGG MAPK Pathway Need: controlled vocabulary for alternative splicing!

14. miR-124 PTB nPTB MEF2 genes KTN1 Additional genes Splicing changes, mediated through CU-rich elements 3’ UTR hexamer Identified through shRNA experiments targeting PTB and nPTB, monitored on microarrays. PTB and nPTB pathway Spellman and Smith, 2006 Boutz et al. Genes Dev. 2007 NeuronsGlial cells Boutz et al. Genes Dev. 2007 (Ares and Black labs)

15. Expression of PTB and nPTB is anti- correlated across human tissues Ratio to reference pool nPTB is enriched in CNS tissues; PTB is depleted. nPTB PTB Brain tissues

16. PTB and nPTB gene expression

17. miR-124 PTB nPTB MEF2 genes KTN1 Additional genes Splicing changes, mediated through CU-rich elements 3’ UTR hexamer Identified through shRNA experiments targeting PTB and nPTB, monitored on microarrays. PTB associated alternative splicing NeuronsGlial cells Boutz et al. Genes Dev. 2007 (Ares and Black labs)

18. Zhu, B. et al. J. Biol. Chem. 2005 MEF2A, MEF2C & MEF2D have a 21 or 24-nt cassette exon, labeled exon β MEF2 transcripts are alternatively spliced; changes lead to increased transcriptional activity Exon β inclusion leads to increased activity in all MEF2 genes.

19. Zhu, B. et al. J. Biol. Chem. 2005 Brain Adipose Heart MEF2A is alternatively spliced in normal tissues Pool The exon is 10-fold upregulated in heart. A junction probe would enable a determination as to whether it is a major form.

20. Brain Adipose Heart Zhu, B. et al. J. Biol. Chem. 2005 MEF2D is alternatively spliced in normal tissues The exon 21-nt long and the associated exon-probe (middle probe) performs poorly. The presence of junction probes permits the measurement.

21. Brain Adipose Heart Zhu, B. et al. J. Biol. Chem. 2005 The mutually exclusive junction probe allows an estimate of the isoform change for exon 8. Change in isoform composition, versus brain: Heart-80 Adipose-82 MEF2C is alternatively spliced in normal tissues

22. Paul L. Boutz et al. Genes Dev. 2007; PTB nPTB MEF2C exon β KTN1 exon 41 40 42 40 41 42 PTB knock-down leads to exon drop PTB and nPTB gene expression correlate with target alternative splice changes across normal tissues

23. PTB levels correlate with KTN1 exon 41 expression

24. PTB associated alternative splicing, motif identification

25. miR-124 transcript targets Transfect miR-124 and microarray profile Lim et al, Nature, 2005 Hexamerp-value gtgcct1.60E-80 tgcctt3.30E-48 agtgcc8.50E-20 gcctta2.90E-17 tgtgcc4.90E-13 gccttt1.30E-10 aagtgc2.80E-10 atgtgc0.0001 ccttac0.003 … gene regulation in our body atlas … 3’ UTR hexamers, using hypergeometric overlap statistics Compare genes downregulated to … miR-124 targets include PTB; which was found down-regulated upon miR-124 transfection and whose 3’ UTR contains 5 hexamers; but not nPTB.

26. Pentamer enrichment adjacent regulated cassette exons cassette exon 3’ pre-mRNA 5’ upstream (udif) upstream intronic fraction (uif) exondownstream intronic fraction (dif) duif dexon5exon3exon5 Motif enrichment in the 200-nt intronic region immediately upstream of cassette exons upregulated in frontal lobe identifies TCTCT. TCTCT CTCTC CTTTC TTTCT CTGCT TGCTT

27. TCTCT enrichment in intronic regions upstream of upregulated cassette exons in human body tissues PTB TCTCT enrichment Upregulation Downregulation - Log10 ratio

28. The intronic region upstream of MEF2A exon β contains a conserved TCTCT

29. TCTCT-associated exons are upregulated with decreasing PTB but not nPTB expression

30. Interpretation of splicing changes Currently in pathways: - transcription factor to targets Add: systems biology at a splicing level - splicing factor to target exons associations - isoform specific nodes in pathways - isoform specific phenotypes & functions PTB MEFC2 w/ exon β KEGG MAPK Pathway Need: controlled vocabulary for alternative splicing

31. Conclusions from PTB story PTB and nPTB expression is anti-correlated across 50 human tissues. PTB expression is anti-correlated with the expression of a set of known cassette exons. The TCTCT pentamer is enriched in the intronic region upstream of the cassette exons. Low PTB expression results in TCTCT enrichment; there is less correlation with nPTB. Pathway interpretation suggested: PTB inhibits expression of cassette exons with TCTCT-rich upstream introns; nPTB is unable to inhibit at least a subset of them

32. Needs for alternative splicing at a pharmaceutical company Biomarker identification Identify transcript structures expressed in a tissue, including novel isoforms Report regulation of known isoforms for use in pathway analysis or as drug targets Requirements Robust, high-confidence, high-throughput profiling –Array patterns, amplification, sequencing Visualization and analysis tools for profiling data Interpretation, including isoform function and pathways

33. Back-ups

34. cassette exon 3’ pre-mRNA 5’ upstream (udif) upstream intronic fraction (uif) exondownstream intronic fraction (dif) duif TCTCT is enriched in the ~50-nt upstream of up- regulated cassette exons Down Up

35. Conclusions from PTB story PTB and nPTB show anti-correlated expression across 50 human tissues. PTB and a set of known cassette exons show anti- correlated expression. The TCTCT hexamer is enriched in the intronic region -70 nt to -30 nt upstream of the cassette exons. Low PTB expression results in TCTCT enrichment; there is less correlation with nPTB. Pathway interpretation suggested: PTB inhibits expression of cassette exons with TCTCT-rich upstream introns; nPTB is unable to inhibit them

36. Novel exon 11 is predicted by a cross- species sequence analysis. These microarray data suggest that the exon is expressed and regulated. Validation of bioinformatics-predicted transcript structures

37. Junction probes enable monitoring of alternative 3’ and 5’ splice sites Sample B Sample A Sample B 1 19 * 20 atg

39. 1 10 * 12 NM_123456 Isoform 2 Cassette Exon Junction probe Exon probe * atg 1 10 * 12 atg 1 10 * 12 atg 1 10 * 12 atg 1 10 * 12 atg 1 10 * 12 atg Some probe strategies for microarray patterns 1 10 * 12 atg All junctions

40. Use of all junction probes reveals potential novel transcript structures Exons 14 and 17 are not previously known to be cassette exons. Exon 3 is a known cassette exon Exon 8 is a known mutually exclusive exon Examine probe intensity (brain) for probes spanning all possible junctions (e.g. exons 1  2, exons 1  3, exons 1  4, …) 1 atg All junctions

41. Bottom line – no predicted changes in alternative splicing, rather a single isoform – of two known - is significantly upregulated. probes w/high predicted background mutually exclusive exon Exon predicted to cross-hyb Sample B Sample A Sample B Sample A 1 10A * atg 10B Mutually exclusive exon pair Real data showing several probes with high background